HAYATI Journal of Biosciences December 2011Vol. 18 No. 4, p 193-196EISSN: 2086-4094

Available online at:http://journal.ipb.ac.id/index.php/hayati

DOI: 10.4308/hjb.18.4.193

SHORT COMMUNICATION

Characterization of Bioflocculant Producing-Bacteria Isolatedfrom Tapioca Waste Water

SURYANI, LAKSMI AMBARSARI, I MADE ARTIKA, HARTUTIK EKA SUSANTI

Department of Biochemistry, Faculty of Mathematics and Natural SciencesDarmaga Campus, Bogor 16680, Indonesia

Received June 7, 2010/Accepted December 15, 2011

Bioflocculant producing-bacteria from tapioca waste water were characterized. Two bacterial isolates i.e. LT-5 andLT-6 isolates had high flocculation activity, with activity of 68.92 and 71.38% respectively. The flocculationactivity of LT-5 isolate increased at pH 2.0-4.0 (acidic condition), however the activity of LT-6 increased at pH 6.0-8.0 (neutral). Addition of 0.05% of AlCl3 as cation was the most effective and had important role in flocculationactivity. Based on the morphological properties, LT-5 isolate was identified as Chromobacterium violaceum and LT-6isolate was identified as Citrobacter koseri.

Key words: bioflocculant producing-bacteria, tapioca wastewater, flocculation activity, cation___________________________________________________________________________

_________________Corresponding author. Phone/Fax: +62-251-8423267,

E-mail: ani3110@yahoo.com

INTRODUCTION

Synthetic flocculant with high molecular weight (suchas polyacrylamide derivatives) have been shown to beharmful to the environment and human health especiallyas neurotoxic and carcinogenic agents of acrylamidemonomers and as source of unbiodegradable pollutants,despite it has effective flocculating performance and lowcost. Because of the negative effect of syntheticflocculant, the used of biodegradable flocculant producedby microorganisms has been investigated in order to findthe biodegradable floculant in widely applications.

Bioflocculant is an extracellular polymer produced bymicroorganisms during their growth, resulting in theformation of stable aggregates of flocs and it has differentcomposition such as protein flocculants, polysaccharideflocculants, glycoprotein flocculants and poly(amino acid)flocculant (Jie et al. 2006). Many studies have beenreported on bioflocculants to replace synthetic flocculantswhich are industrially used. Bioflocculant may potentiallybe applied in drinking and wastewater treatment,downstream processing, and fermentation processes(Salehizadeh & Shojaosadati 2001). Kurane and Nohata(1997) found that bioflocculants have widebroad spectrumto be applied in industries. They found that bioflocculantsproduced by Alcaligenes latus can absorb water 1000time of its weight and 5 time stronger than that of syntheticabsorber polymers.

In recent years, a major emphasis has been laid on thesearch for novel bioflocculant producing-bacteria with

different composition and properties and several of themhave been under investigated. The use of bioflocculantsis very prospective in the future, therefore it is importantto explore potential bioflcoculant producing-bacteria withhigh activity bioflocculant-producing ability and improvethe flocculating activity. In this study, the screening of abioflocculant producing-bacteria and optimization of theflocculation activity were carried out.

MATERIALS AND METHODS

Isolation Bioflocculants Producing Bacteria. Sampleswere taken from tapioca waste water at Sukaraja region,Bogor, West Java, Indonesia. Isolation of bacteria wasdone according to general method for microbe isolationby using NB medium. One ml of samples were cultivatedin 9 ml of NB medium and incubated at 27 oC for 16 hours.The samples were serially diluted and 0.1 ml was platedonto petri dish. After 16 hours incubation, bacterialcolonies were counted and grown in NB medium.

Bioflocculants Production. Pure bacterial isolates wereinoculated into 30 ml of production medium. Compositionof the medium was 10 g of glucose, 10 g of sucrose, 0.5 gof peptone, 0.5 g of yeast extract, 1 g of urea, 0.5 g(NH4)2SO4, 1.5 g KH2PO4, 4.5 g of K2HPO4, 0.2 g MgSO4,and 0.1 g of NaCl in l l of sterile aquades and the pH wasadjusted to 7.3 (Kurane et al. 1997). The cultures wereincubated in rotary shaker at 27 oC with 120 rpm for70 hours. The bacterial cultures were used fordetermination of flocculation activity.

Determination of Flocculation Activity. Theflocculation activity was determined by optimization of

coagulant and flocculation activity assay in kaolinsuspension (Kurane et al. 1997). Optimization of coagulantwas done by using FeCl3 with dosage range between 0.01-1.0% b/v. The solution was mixed gently at roomtemperature (27 oC) and left for standing in 5 minutes. Theformation of visible aggregates was observed andmeasured its absorbance at wavelength of 550 nm.Flocculation activity assay was done in 80 ml of kaolinsuspension (5.5 g/l) and mixed with the optimum of FeCl3dosage solution and 1 ml of culture, then adjusted to100 ml with sterile aquades. The solution was mixed gentlyat room temperature (27 oC) and left for standing in 5 minutes.The optical density (OD) was measured at 550 nm.Selection of bioflcoculant producing-bacteria based onthe highest activity of flocculation. Flocculation activitywas calculated by the following equation:

Flocculation activity = [A-B/A] x 100%where: A = OD550 of reference, B = OD550 of sample

Optimization of Flocculation Activity. Optimization offlocculation activity was conducted according to Kuraneet al. 1997 with modification on cation addition, pH andtemperature. Effect of cation addition were done in 80 mlof kaolin suspension (5.5 g/l), 1 ml of bacterial culture andseveral salt solution (0.05%) as cation sources i.e. CaCl2,MgSO4, AlCl3, FeCl3, FeSO4, dan ZnSO4. The effect of pHin kaolin suspension was determined at pH range of 2.0-9.0. The effect of pH on flocculation activity was done attemperature of 30, 40, 50, 60, and 70 oC. In this assay, thebacterial cultures were incubated for 30 minutes and theflocculation activity was determined as describedpreviously.

Bacterial Identification. Identification of bioflocculantproducing-bacteria was done for 2 selected isolates atIndonesian Research Center for Veterinary Sciences/BALITVET).

RESULTS

Flocculation Activity of Bioflocculants Producing-Bacteria. Temperature and of sampling sites was 27.5 oC

and pH 7.0 respectively. Eights isolates of bioflocculantsproducing-bacteria were isolated from the waste watertapioca (Table 1). Production of bioflocculant was occurredat stationary phase after 70 hours incubation on a rotaryshaker at 120 rpm (unpublished data). Addition of substratein growth medium affected the bacterial growth andbioflocculant production.

Flocculation Activity in Kaolin Suspension. Based onoptimization of FeCl3 dosage as coagulant, the optimumdosage was 0.05% b/v of FeCl3 which had the highestflocculation activity (55.47%) compared to otherconcentrations (Table 2). Flocculation activity increasedup to 55.47% at 0.01-0.05%. There was no flocculationactivity observed at 1% of FeCl3. The ability of each isolateshowed different flocculation activity (Table 1). Thehighest activity of flocculation activity was shown byLT-5 and LT-6 isolates with the flocculation activity up to68.92 and 71.38%, respectively. Therefore, LT-5 and LT-6isolates were selected for further investigation(optimization of flocculation activity).

Optimum Condition of Flocculation Activity. Bacterialisolate of LT-5 gave optimum activity when AlCl3 was usedas cation with activity of 30.76%, whereas LT-6 isolategave the optimum activity when AlCl3 and FeCl3 were usedas cation with activity of 62.29 and 54.12%, respectively(Figure 1). Cation addition gives the effect significantlyon flocculation activity for all isolates in kaolin suspension.Optimum flocculation activity was found on addition ofAlCl3 0.03%.

The result showed that pH had significant effect onflocculation activity. Each isolate had different optimumpH for flocculation activity (Figure 2). Flocculation activityof LT-5 isolate was increased at pH 2.0 up to 4.0 (acidiccondition) with flocculation activity up to 40.47%.However, LT-6 isolate had optimum pH in the range of 6.0-8.0 (neutral), and the highest flocculation activity was atpH 6.0 (42.43%).

Temperature effect on flocculation activity showedthat LT-5 isolate had optimum temperature at 40 oC withflocculation activity up to 73.11%, whereas LT-6 isolate

Table 2. Optimization of FeCl3 dosage as coagulant

Dosage of FeCl3 (%) Rate of floc formation Optical density of supernatant Flocculation activity (%)0.0100.0250.0500.1001.000

30.4846.9255.4729.79

-24.70

++++++++

++

0.2030.1550.1300.2050.363

+ = low, ++ = medium, +++ = high.

Table 1. Morphological colony and their flocculation activity of bacterial isolates isolated from tapioca waste water

Isolate Color/shape Rate of flocculation formation Flocculation activity (%)LT-1LT-2LT-4LT-5LT-6LT-9LT-11LT-12

47.0762.7758.4668.9271.3861.2313.5450.46

White/round plateYellow/roundYellow/rootyBlue/rootyYellow greenishGreenLight yellowWhite/round thick

++++

++++++++-+

- = none, + = low, ++ = medium, +++ = high.

194 SURYANI ET AL. HAYATI J Biosci

had optimum temperature at 30 oC with flocculationactivity up to 75.94% (Figure 3). Flocculation activity forLT-6 isolate was decreased slightly at 40 oC comparedwith at 30 oC. Flocculation activity at 40 oC was 73.58%and this flocculation activity was the same value withLT-5 isolate.

Bacterial Identification. Identification result showedthat LT-5 isolate was Chromobacterium violaceum andLT-6 isolate was Citrobacter koseri.

DISCUSSION

Characterization of bioflocculant producing-bacteriawas carried out from the waste water tapioca. Thewastewater tapioca contained soluble starch and otherorganic compounds as carbon sources. This conditioncan promote the growth of bioflocculant producing-bacteria. The starch can be modified into a flocculantthrough chemical reaction (Khalil & Aly 2001). Throughthe action of bacteria, starch can be easily changed intoan effective polysaccharide or glycoproteinbioflocculants. Therefore, the bioflocculant producing-bacteria from the waste tapioca can grow easily. Yue (2006)found on Klebsiella sp. that high floculating activity ofthe culture containing soluble starch mainly came fromsoluble starch which is also a polysaccharide. Solublestarch was the most suitable agent for bioflocculant, theflocculating rate of 0.5 ml of the sterile medium containingsoluble starch was 70.1%. Zhang et al. 2002 found thatthe optimum carbon source for Sorangium cellulosumwas soluble starch and 14.8 gl-1 polysaccharidebiocflocculant can be produced in the medium containing30 gl-1 soluble starch.

Production of bioflocculant has been carried out onmedium containing glucose and sucrose as the maincarbon source. Both of these sugars appeared favorablefor cell growth as well as for bioflocculant production.The combination of urea and (NH4)2SO4 as inorganic ofnitrogen sources enhanced the cell activity to producebioflocculant. Production of bioflocculant was carried outfor 70 hours at 120 rpm. Jie et al. 2006 reported that cells ofVagococcus sp. W13 grew rapidly in the first 60 hours ofcultivation, and then leveled off. Production ofbioflocculant occurs at the early of exponential phasethen it is increased up to the optimum condition before itis decreased (Sumarno 2000) causing the bioflocculantswere changed into others products by some enzymes ofbacteria. Kurane et al. (1986) observed that the maximumbioflocculant formation was in the early stationary phase,and no rapid decrease of flocculating activity was observedin late stationary phase.

Flocculation activity was affected by cation, pH, andtemperature. The concentration of FeCl3 as coagulant wasdetermined in order to obtain the optimum concentrationfor flocculation activity assay, since for each assay thereis no specific coagulant and its concentration (Parwono1998). Flocculation activity was different for each isolate.The formation of floc is due to the binding ofbioflocculants and FeCl3 0.05% to form colloid.Flocculation process produces bigger particles andaccelerates precipitation. Bigger form of aggregate willaffect formation of floculated kaolin clay.

Flocculation activity assay requires the cation ascoagulant. Both LT-5 and LT-6 isolates gave the highestflocculation activity by adding FeCl3 and AlCl3 as

0

5

10

15

20

25

30

35

40

45

2 3 4 5 6 7 8 9pH

Floc

cula

tion

activ

ity (%

)

Figure 2. Effect of pH on flocculation activity of bioflocculantfrom LT-5 and LT-6 isolates. : LT-5, : LT-6.

0

10

20

30

40

50

60

70

80

30 40 50 60 70Temperature (oC)

Floc

cula

tion

activ

ity (

%)

Figure 3. Effect of temparature on flocculation activity ofbioflocculant from LT-5 and LT-6 isolates. : LT-5, : LT-6.

0

10

20

30

40

50

60

70

FeCl3 ZnSO4 MgSO4 CaCl2 FeSO4 AlCl3Cation

Floc

cula

tion

activ

ity (

%)

Figure 1. Effect of cation addition on flocculation activity ofbioflocculant from LT-5 and LT-6 isolates. : LT-5, :LT-6.

Vol. 18, 2011 SHORT COMMUNICATION 195

coagulant. It is assumed that Fe3+ ion stimulate flocculationby neutralization and stabilization of residual negativecharges of carboxyl groups of uronic acid in acidicpolysaccharides, forming bridges which bind kaolinparticles to each other (Yokoi 1998). The positive chargesof FeCl3 will uptake the negative charges of particles. Thiscondition will cause the interaction between colloid andformed larger floc, it is usually called coagulation process.FeCl3 as a cation source was used in this assay. FeCl3 hastrivalent and big positive charge. Therefore it has abilityto replace negative charge in the colloid and reduce thebarrier energy. Cation has important role to stimulatecoagulation process in kaolin suspension. The additionof cation to the reaction mixture was necessary to induceeffectively flocculation by forming complexes ofpolysaccharides and kaolin clay mediated by a cation(Kurane & Matsuyama 1994). The synergistic effects oftrivalent cations were stronger than that of bivalentcations, with Al3+ being the most effective cation (Toeda1991). Addition of cation gave significant effect to thereaction mixture on flocculation activity of LT-5 and LT-6isolates in kaolin suspension. Optimum of flocculationactivity was reached by adding 0.05% of AlCl3. The resultsproved that bioflocculant has ability to form agregationwith alumunium and increase flocculation effectivity.

The pH reaction is known to be a key factor influencingflocculating activity (Yokoi et al. 1996). This study showedthat there was a significant effect of pH on flocculationactivity (Figure 2). Different response of flocculationactivity on LT-5 and LT-6 isolates to various pH describedthe difference of isoelectric point for each isolate.Isoelectric point is requirements for flocculation andcoagulation process. Zhang et al. (2002) found thatflocculation activity of crude exopolysaccharide from S.cellulosum was correlated to the pH. Bioflocculant p-KG03was reported to be active at acidic conditions (pH 3-6)(Yim et al. 2007), while bioflocculant of Nannocystis sp.NU-2 was active in alkaline conditions (pH 12-14) in thepresence of 30 mg/l CaCl22H2O (Zhang et al. 2002).According to Jie et al. (2007) either using broth culture ofVagococcus sp. W13 or purified flocculant, the flocculatingactivity was high and stable at pH 7.0-10.0. The LT-5 andLT-6 isolates showed flocculation activity at pH 2.0-4.0(40.47%) and pH 6.0-8.0 (42.43%), respectively. It is showedthat each isolates has different optimum pH forflocculation. Most reports indicated that pH affected theflocculating efficiency of bioflocculant. According toKurane et al. (1994), little flocculation occurred in the pHrange above 8.0. It suggested that the property of LT-5and LT-6 isolates may facilitate its application in variouspH conditions of waste-water environments.

Optimum temperature is needed to obtain the maximumflocculation activity. Based on this study, the increasingof temperature affected to decreasing of flocculationactivity with average about 34% for LT-5 isolate and 33%for LT-6 isolate. However, flocculation acivity for bothisolates was still observed at 50, 60, and 70 oC after30 minutes incubation. Average of flocculation activityof LT-5 and LT-6 isolates was 48 and 49% respectively.Based on the data, it can be concluded that LT-5 and LT-6

isolates had optimum temperature for flocculation activityat 40 oC.

ACKNOWLEDGEMENT

We thank to Directorate General of Higher Educationfor this research funding. We would like to acknowledgeDepartment of Biochemistry, Faculty Mathematics andNatural Sciences, Bogor Agricultural University forproviding the laboratory facilities, and IndonesianResearch Center for Veterinary Sciences/BALITVET forbacterial identification assistance.

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196 SURYANI ET AL. HAYATI J Biosci